Department of Women’s and Children’s Health Department of Women’s and Children’s Health Karolinska Institutet, Stockholm, Sweden Karolinska Institutet, Stockholm, Sweden ROLE OF INTERMEDIATE FILAMENT ROLE OF INTERMEDIATE FILAMENT DESMIN IN DEVELOPMENT OF DESMIN- DESMIN IN DEVELOPMENT OF DESMIN- RELATED MYOPATHY RELATED MYOPATHY Natalia Smolina Natalia Smolina Stockholm 2015 Stockholm 2015 All previously published papers were reproduced with permission from the All previously published papers were reproduced with permission from the publisher. publisher. Published by Karolinska Institutet. Published by Karolinska Institutet. Printed by E-print AB 2015. Printed by E-print AB 2015. © Natalia Smolina, 2015 © Natalia Smolina, 2015 ISBN 978-91-7676-065-9 ISBN 978-91-7676-065-9 ROLE OF INTERMEDIATE FILAMENT DESMIN IN ROLE OF INTERMEDIATE FILAMENT DESMIN IN DEVELOPMENT OF DESMIN-RELATED MYOPATHY DEVELOPMENT OF DESMIN-RELATED MYOPATHY THESIS FOR DOCTORAL DEGREE (Ph.D.) THESIS FOR DOCTORAL DEGREE (Ph.D.) By By Natalia Smolina Natalia Smolina Principal Supervisor: Opponent: Principal Supervisor: Opponent: Professor Thomas Sejersen Professor Oliver Friedrich Professor Thomas Sejersen Professor Oliver Friedrich Karolinska Institutet Friedrich-Alexander-University Karolinska Institutet Friedrich-Alexander-University Department of Women’s and Children’s Health Erlangen-Nuremberg Department of Women’s and Children’s Health Erlangen-Nuremberg Division of Pediatric Neurology Institute of Medical Biotechnology Division of Pediatric Neurology Institute of Medical Biotechnology Co-supervisors: Examination Board: Co-supervisors: Examination Board: Dr. Gunnar Sjoberg Professor Lars Larsson Dr. Gunnar Sjoberg Professor Lars Larsson Karolinska Institutet Karolinska Institutet Karolinska Institutet Karolinska Institutet Department of Women’s and Children’s Health Department of Physiology and Pharmacology Department of Women’s and Children’s Health Department of Physiology and Pharmacology Division of Pediatric Cardiology Division of Pediatric Cardiology Associate professor Erik Björck Associate professor Erik Björck Dr. Anna Kostareva Karolinska Institutet Dr. Anna Kostareva Karolinska Institutet Karolinska Institutet Department of Molecular Medicine and Surgery Karolinska Institutet Department of Molecular Medicine and Surgery Department of Women’s and Children’s Health Division of Clinical Genetics Department of Women’s and Children’s Health Division of Clinical Genetics Division of Pediatric Neurology Division of Pediatric Neurology Docent Cecilia Gunnarsson Docent Cecilia Gunnarsson Linköping University Linköping University Department of Clinical and Experimental Department of Clinical and Experimental Medicine Medicine Division of Cell Biology Division of Cell Biology To my Babusya and Dedusya To my Babusya and Dedusya ABSTRACT ABSTRACT Desmin is a major intermediate filament of muscle cells, serving to transmit Desmin is a major intermediate filament of muscle cells, serving to transmit mechanical forces and propagate mechanochemical signals, to coordinate mechanical forces and propagate mechanochemical signals, to coordinate contraction and relaxation cycles, and to stabilize the positioning of cellular contraction and relaxation cycles, and to stabilize the positioning of cellular organelles, e.g. mitochondria. Around 70 desmin gene mutations have been organelles, e.g. mitochondria. Around 70 desmin gene mutations have been reported in conjunction with desmin-related myopathy. Desmin-related myopathy reported in conjunction with desmin-related myopathy. Desmin-related myopathy can be described as pathophysiological complex, accompanied by desmin can be described as pathophysiological complex, accompanied by desmin intracellular aggregate accumulation and impairment of desmin interactions with intracellular aggregate accumulation and impairment of desmin interactions with structural proteins, signal molecules, and cell organelles. However, the precise structural proteins, signal molecules, and cell organelles. However, the precise molecular mechanism underlying desmin-related myopathy have not been molecular mechanism underlying desmin-related myopathy have not been described yet. There are speculations if it is connected with toxic effects of desmin described yet. There are speculations if it is connected with toxic effects of desmin aggregates or with violation of desmin mechanotransduction functions. aggregates or with violation of desmin mechanotransduction functions. The general aim of the present PhD project was to extend existing knowledge about The general aim of the present PhD project was to extend existing knowledge about the molecular machinery on how desmin gene mutations lead to the development the molecular machinery on how desmin gene mutations lead to the development of desmin-related myopathy, with an emphasis on development of of desmin-related myopathy, with an emphasis on development of cardiomyopathies. To address this aim the following research questions were cardiomyopathies. To address this aim the following research questions were stated: (i) genetic study of a group of patients with cardiomyopathies in order to stated: (i) genetic study of a group of patients with cardiomyopathies in order to describe novel mutations in the desmin gene, and to assess the frequency of DES describe novel mutations in the desmin gene, and to assess the frequency of DES A213V; (ii) genetic study by means of next-generation sequencing approach of a A213V; (ii) genetic study by means of next-generation sequencing approach of a group of patients with idiopathic restrictive cardiomyopathy in order to describe group of patients with idiopathic restrictive cardiomyopathy in order to describe novel genetic variants associated with disease; (iii) functional study of desmin gene novel genetic variants associated with disease; (iii) functional study of desmin gene point mutations effect on mitochondrial properties. point mutations effect on mitochondrial properties. The main findings regarding genetic background were: (i) DES A213V represents a The main findings regarding genetic background were: (i) DES A213V represents a disease-modifying polymorphism, rather than disease-related mutation, since it disease-modifying polymorphism, rather than disease-related mutation, since it was found both in patients and healthy donors; (ii) combination of disease-related– was found both in patients and healthy donors; (ii) combination of disease-related– disease-modifying or disease-related–disease-related genetic variants, rather than disease-modifying or disease-related–disease-related genetic variants, rather than single disease-related mutation, determined the development of idiopathic single disease-related mutation, determined the development of idiopathic restrictive cardiomyopathy. Most proteins of these combinations belonged to four restrictive cardiomyopathy. Most proteins of these combinations belonged to four functional groups: sarcomeric contractile proteins, mechanosensing Z-disc functional groups: sarcomeric contractile proteins, mechanosensing Z-disc proteins, nuclear membrane, and outer mitochondrial membrane proteins. proteins, nuclear membrane, and outer mitochondrial membrane proteins. Functional studies of the impact of desmin mutations on mitochondria showed that Functional studies of the impact of desmin mutations on mitochondria showed that aggregate-prone mutations decreased mitochondrial calcium uptake, as well as aggregate-prone mutations decreased mitochondrial calcium uptake, as well as depressed maximal oxygen consumption rate and spare respiratory capacity. In depressed maximal oxygen consumption rate and spare respiratory capacity. In contrast, non-aggregate-prone mutations did not disturb mitochondrial calcium. contrast, non-aggregate-prone mutations did not disturb mitochondrial calcium. They did, however, result in the reduction of maximal oxygen consumption rate They did, however, result in the reduction of maximal oxygen consumption rate and affected spare respiratory capacity. and affected spare respiratory capacity. To conclude, (i) distortion of desmin mechanotransduction functions plays an To conclude, (i) distortion of desmin mechanotransduction functions plays an important role in desmin-related myopathy onset, affecting mitochondrial important role in desmin-related myopathy onset, affecting mitochondrial properties; (ii) combination of mutations in genes encoding sarcomeric contractile properties; (ii) combination of mutations in genes encoding sarcomeric contractile and mechanosensing proteins, rather than a single mutation, predisposes to the and mechanosensing proteins, rather than a single mutation, predisposes to the development of cardiomyopathy. These data facilitate understanding of molecular development of cardiomyopathy. These data facilitate understanding of molecular pathways underlying desmin-related myopathy development, and increase existing pathways underlying desmin-related myopathy development, and increase existing knowledge of intracellular interactions within the muscle cell.!! knowledge of intracellular interactions within the muscle cell.!! LIST OF SCIENTIFIC PAPERS LIST OF SCIENTIFIC PAPERS I. Gudkova A, Kostareva A, Sjoberg G, Smolina N, Turalschuk M, I. Gudkova A, Kostareva A, Sjoberg G, Smolina N, Turalschuk M, Kuznetsova I, Rybakova M, Edstrom L, Shlyakhto E, Sejersen T. Kuznetsova I, Rybakova M, Edstrom L, Shlyakhto E, Sejersen T. Diagnostic challenge in desmin cardiomyopathy with